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In JoVE (1)
Other Publications (10)
- Neuroscience Research
- Cell and Tissue Research
- The Biochemical Journal
- The Journal of Physical Chemistry. B
- Journal of Biological Physics
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Lab on a Chip
- The Journal of Biological Chemistry
- The Review of Scientific Instruments
- Biophysical Journal
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Articles by Valerica Raicu in JoVE
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In vivo Kvantifiering av G Tillsammans Interaktioner protein receptor med spektralt korrigerats inom två-photon Mikroskopi
1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee
Genom att använda ett spektralt löst två-photon-systemet mikroskopi avbildning, är pixelnivå kartor över Förster Resonance Energy Transfer (FRET) effektivitetsvinster för celler som uttrycker membranreceptorer hypoteser för att bilda homo-oligomeric komplex. Från FRET effektivitet kartor, kan vi uppskatta stökiometriska information om oligomer komplex under utredning.
Other articles by Valerica Raicu on PubMed
Combined Argatroban and Edaravone Caused Additive Neuroprotection Against 15 Min of Forebrain Ischemia in Gerbils
We investigated whether or not a combination of the selective thrombin inhibitor, argatroban, and the free radical scavenger, edaravone (MCI-186), ameliorates postischemic hypoperfusion and decreases mortality after 15 min of forebrain ischemia in the gerbil. Argatroban or edaravone alone significantly increased postischemic cerebral blood flow and attenuated brain edema after reperfusion. However, only the combination increased the survival ratio (P<0.05 by Mantel-Cox) and protected the damage of neuronal cells. The present study indicates that anticoagulants and free radical scavengers reciprocally function to inhibit the progression of ischemic cell damage and that a combination of these types of drugs will help to improve the outcomes after cerebral ischemia.
Acute Ischemia Causes 'dark Cell' Change of Strial Marginal Cells in Gerbil Cochlea
The cochlear stria vascularis produces the endolymph and generates the endocochlear DC potential, two indispensable ingredients of an auditory transduction process. The marginal cell, one of the several cell types constituting the stria vascularis, is called 'the dark cell' on the basis of its appearance by transmission electron microscopy (TEM). To clarify whether this commonly observed 'dark appearance' is a normal characteristic of marginal cells, as conjectured in the literature, or an experimental artifact, we developed an in vivo fixation method for minimizing ischemic tissue damages. While under sustained systemic circulation with oxygenated blood, the stria vascularis of gerbils was chemically fixed by perilymphatic perfusion with a fixative, and the stria vascularis was observed by TEM. In contrast to a number of previous reports, the cytoplasm of marginal cells was not dark, and quantitative analysis showed that the difference between the cytoplasmic electron density of marginal cells and that of intermediate cells (another type of strial cells) was not statistically significant. For comparison, the gerbils were allowed to undergo 3 min of ischemia following decapitation. Under these conditions, marginal cells showed typical 'dark appearance', as reported previously, and their cytoplasmic electron density was 1.7 times higher than that of the intermediate cells. In addition, the volume of mitochondria in marginal cells undergoing 3 min of ischemia was higher than that fixed in vivo. We therefore conclude that the widely recognized 'dark cell' appearance of marginal cells following conventional fixation procedures reflects cell injury due to ischemia, which is inherent in the standard fixation procedures, but can be avoided by our fixation protocol here introduced.
Protein Interaction Quantified in Vivo by Spectrally Resolved Fluorescence Resonance Energy Transfer
We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.
Determination of the Fe-CO Bond Energy in Myoglobin Using Heterodyne-detected Transient Thermal Phase Grating Spectroscopy
The bond energies at active sites of proteins are intimately coupled to the structure-function relationship in biological systems. Due to the unknown nature of the protein relaxation along a reaction coordinate, it has not been possible to directly determine bond energies relevant to protein function. By embedding proteins in trehalose glasses, it is possible to freeze out protein relaxation on short time scales and determine the bond energies of photolabile ligands using photothermal spectroscopies. As a prototypical example, the photodissociation dynamics and energetics of carboxy-myoglobin (MbCO) in a trehalose glass matrix at room temperature were studied by transient absorption (or pump-probe) and transient thermal phase grating spectroscopy to determine the CO recombination dynamics and associated energetics, respectively. Both the initial energetics of the bond breaking and the energy released upon bond reformation could be used, on a time scale faster than significant protein relaxation, to determine the Fe-CO bond energy as 34 +/- 4 kcal/mol. This bond energy is significantly larger than that typically cited (25 kcal/mol) on the basis of indirect measurements but is in good agreement with recent theoretical predictions (35 kcal/mol) (Rovira, C.; Parrinello, M. Int. J. Quantum Chem. 2000, 80, 1172). This result in combination with the theoretical study suggests that protein structure plays a significant role in the bond energies at active sites which in turn provides a tuning element of the effective barrier heights independent to the transition state region.
Efficiency of Resonance Energy Transfer in Homo-oligomeric Complexes of Proteins
A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.
Implementation of a Fast Reconfigurable Array for Tissue Impedance Characterization
Various tissue properties have been used in the past and present as metrics which can serve to discriminate healthy from diseased tissue. Electromagnetic absorption (of x-rays and optical signals), scattering of near-infrared light, and electrical impedance are a few such parameters. In order to serve as discriminants for diseased (e.g., neoplastic) tissue, the characteristics of these tissues must first be precisely determined. In this paper, we consider the electrical impedance properties of tissues and cell aggregates, and present the design of a reconfigurable electrode array which is capable of providing a well-defined electromagnetic interface to the tissue under study, for characterization in the 0.01-30 MHz range. The configuration of array elements may be easily changed under digital control, allowing for various electromagnetic field configurations to be applied to the tissue under study. The array is designed to interface to four-point as well as two-point impedance instrumentation, and may be used for two-dimensional bioimaging systems based on electrical impedances. The design may be scaled to higher frequencies and smaller dimensions, allowing for studies of electrical properties at the cellular level.
Real-time Monitoring of Two-photon Photopolymerization for Use in Fabrication of Microfluidic Devices
We report an improved method for production of microfluidic device masters using two-photon photopolymerization of SU-8 negative photoresist, which relies on a two-photon microscope (TPM) commonly used in imaging of biological samples. The device masters serve as negative relief structures for polydimethylsiloxane-based microfluidic devices. We observed that not only did the two-photon excitation of the SU-8 photoresist initiate crosslinking of the material in the region of the focus of the near-infrared laser beam (as expected) but it also resulted in emission of fluorescence in the visible range. The detected emission of SU-8 photoresist undergoing two-photon excitation displayed a strong correlation with the size of the polymerized objects produced during the exposure; this allowed the progress of the microfluidic master production process to be monitored in real-time. We demonstrate the use of the fluorescence detection during two-photon photopolymerization in the production of microfluidic devices, which were designed to trap individual yeast cells to be imaged with the same TPM used for microfluidic master writing.
Oligomeric Size of the M2 Muscarinic Receptor in Live Cells As Determined by Quantitative Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M(2) muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M(2) receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M(2) receptor to enhanced green fluorescent protein-M(2) receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 +/- 0.019; n = 4, 0.202 +/- 0.010; n = 6, 0.128 +/- 0.006; n = 8, 0.093 +/- 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20-0.24, identifying the M(2) receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.
Two-dimensional Dielectric Spectroscopy: Implementation and Validation of a Scanning Open-ended Coaxial Probe
Dielectric spectroscopy is a powerful tool for characterizing and classifying materials based on their electrical properties. In order to perform dielectric measurements on a sample with spatially varying properties, the measuring probe typically is repositioned manually on the surface of the sample for each measurement. In this paper, we present a novel technique, based on a reconfigurable multielectrode array, which facilitates the recording of measurements at various different spatial locations without physically moving the measuring electrodes. By electronically selecting one of the electrodes as the inner line and connecting the remainder of the electrodes together to form the outer line, an open-ended coaxial probe is created, which can be repositioned by simply selecting different electrode combinations; hence the name of a "traveling" coaxial probe. The geometric factor, or the cell constant, of each coaxial probe in the array was estimated from measurements on saline solutions with known electrical characteristics. In order to validate the setup for measurement of dielectric properties of biological cells, the plasma membrane capacitance and cytoplasm conductivity of yeast cells suspended in aqueous solutions were measured and compared to results from published reports. Dielectric spectroscopy imaging was carried out on tissue phantoms made of an agar gel with inclusions consisting of concentrated yeast cell suspensions. Measurements were performed on the phantoms, and the dielectric data were spatially mapped with respect to electrode location. The spatial electrical data correlated precisely with locations of yeast cell inclusions within the phantoms.
Comparison Between Whole Distribution- and Average-based Approaches to the Determination of Fluorescence Resonance Energy Transfer Efficiency in Ensembles of Proteins in Living Cells
Current methods for analysis of data from studies of protein-protein interactions using fluorescence resonance energy transfer (FRET) emerged from several decades of research using wide-field microscopes and spectrofluorometers to measure fluorescence from individual cells or cell populations. Inherent to most measurements is an averaging of the distributions of FRET efficiencies over large populations of protein complexes, which washes out information regarding the stoichiometry and structure of protein complexes. Although the introduction of laser-scanning microscopes in principle could facilitate quantification of the distributions of FRET efficiencies in live cells, only comparatively recently did this potential fully materialize, through development of spectral- or lifetime-based approaches. To exploit this new opportunity in molecular imaging, it is necessary to further develop theoretical models and methods of data analysis. Using Monte Carlo simulations, we investigated FRET in homogenous and inhomogeneous spatial distributions of molecules. Our results indicate that an analysis based on distributions of FRET efficiencies presents significant advantages over the average-based approach, which include allowing for proper identification of biologically relevant FRET. This study provides insights into the effect of molecular crowding on FRET, and it offers a basis for information extraction from distributions of FRET efficiencies using simulations-based data fitting.
